Increased recovery of active proteins

ABSTRACT

The invention provides methods of increasing yields of desired conformation of proteins. In particular embodiments, the invention includes contacting preparations of a recombinant protein with a reduction/oxidation coupling reagent for a time sufficient to increase the relative proportion of a desired configurational isomer.

This application claims the benefit of provisional U.S. application60/271,033, filed Feb. 23, 2001, the disclosure of which is incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The invention is in the field of treatment and purification of proteins.

BACKGROUND

High levels of expression of many proteins of eukaryotic origin havebeen achieved in prokaryotic expression hosts. Such eukaryotic proteinsoften misfold and accumulate as insoluble inclusion bodies in theprokaryotic host. In order to obtain biologically active protein, theproteins trapped in inclusion bodies had to be unfolded and refoldedunder harsh conditions including chaotropic agents and reducing thiols.

Expression of proteins of eukaryotic origin in eukaryotic hosts avoidedthese problems. Provided that the expression vector was properlydesigned (e.g., with secretory signal peptides, etc.), eukaryotic celllines tend to correctly process and secrete extracellular eukaryoticproteins as soluble products.

However, as expression systems and vectors have been improved tomaximize levels of expression from eukaryotic hosts, not all of therecombinant protein expressed and secreted from these hosts is in thedesired, most active conformation. The invention is designed to overcomesuch expression problems, and maximize yields of biologically activeprotein.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that not all of thepreparation of recombinant protein that is expressed by eukaryotic hostcells is folded into a native tertiary conformation. In addition, it hasbeen found that regions or domains of recombinant proteins may beproperly folded, while other regions or domains may have undesiredconformations. Accordingly, in one aspect, the invention provides amethod of contacting a preparation of the recombinant protein thatcontains a mixture of at least two isomers of the recombinant protein toa reduction/oxidation coupling reagent for a time sufficient to increasethe relative proportion of the desired conformational isomer anddetermining the relative proportion of the desired conformational isomerin the mixture. In another aspect, the invention entails contacting apreparation of a recombinant protein that has been produced by mammaliancells with a reduction/oxidation coupling reagent, at a pH of about 7 toabout 11, and isolating a fraction of the preparation of the recombinantprotein with a desired conformation. Preferred recombinant proteins areglycosylated recombinant proteins such as, e.g., those produced byeukaryotic cells. The invention also relates to methods of formulatingthe resulting preparations into a sterile unit dose form, andcompositions produced by the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Hydrophobic interaction chromatography (HIC) of TNFR:Fc. Thispreparation of TNFR:Fc elutes during HIC as three distinct peakscollected into Fraction #2 and Fraction #3, as indicated.

FIG. 2. Circular Dichroism Analysis of Fractions #2 and #3. Near-UVCircular Dichroism measurements expressed in terms of mean residueellipticity are shown in FIG. 2. FIG. 2A presents the spectral data; Theline for Fraction #3 is closest to the arrow highlighting the negativedisplacement at about 270 nM ascribed to disulfide contributions, andthe line for Fraction #2 is the darker solid line. FIG. 2B presents thecurve-fitted data for Fraction #2 (small dashed line) and Fraction #3(larger dashed line).

FIG. 3. Molecular Weight Determination Using On-line size exclusionchromatography (SEC), ultraviolet (UV), light scattering (LS), andrefractive index (RI) detection in series (On-line SEC/UV/LS/RI). FIG.3A is Fraction #3, and FIG. 3B is Fraction #2. Vertical dashed linesindicate where the slices were evaluated for molecular weightdetermination in the region surrounding the main peak.

FIG. 4. Differential Scanning Calorimetry Analysis of Fractions #2 and#3. FIG. 4A is the uncorrected data, and FIG. 4B presents thebaseline-corrected data. Thermal melting transitions are labeled byvertical dashed lines. Arrows indicate an enthalpy displacement. Thehorizontal dotted lines in FIG. 4B are used as a baseline reference.

FIG. 5. Correlation of Fraction #2 and Binding Activity. Six differentpreparations of TNFR:Fc (denoted A through F), from six different celllines, were tested for the correlation between the percent increase inproportion of Fraction #2 (dark diamonds) and percent increase in TNFalpha Binding Units (light diamonds).

FIG. 6. Effect of Varying Cysteine Concentration on Conversion ofFraction #3 into Fraction #2. Protein samples were treated with variousconcentrations of cysteine (0.25–5.0 mM) and changes in Fraction #3assessed using HIC. Four different lots of TNFR:Fc were treated for 18hours at the indicated cysteine concentration on the x-axis. The percentof Fraction #3 in each lot that was converted into Fraction #2 isplotted on the y-axis.

FIG. 7. Effect of Cysteine Concentration on Proportion of Fraction #3.Protein samples from four different lots were treated with variousconcentrations of cysteine (0–50 mM) and the resulting level of Fraction#3 was assessed by HIC.

FIG. 8. Effect of Temperature on Disulfide Exchange. Protein fractionswere treated at room temperature or 4 degrees C. in the presence orabsence of copper for various times. FIG. 8A presents changes in HICFraction #3 after 6 Hours, and FIG. 8B presents changes in HIC Fraction#3 after 18 hours.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of increasing the recovery of activerecombinant proteins. In particular, the invention involves promoting adesired conformation of a protein in preparations of a recombinantprotein. Significantly, the invention provides gentle methods ofaltering protein structure without necessitating the use of harshchaotrope treatments (such as, for example, strong denaturants such asSDS, guanidium hydrochloride or urea). Using the methods of theinvention on preparations of recombinant protein results in a higherpercentage, or higher relative fraction, of the recombinant protein inthe preparation with a desired conformation. A desired conformation fora recombinant protein is the three-dimensional structure of a proteinthat most closely resembles, and/or duplicates the function of, thenaturally occurring domain of that protein. Such gentle methods areparticularly advantageous when the recombinant protein is intended to beused in vivo as a drug or biologic.

Generally, when the recombinant protein contains a domain of a receptorprotein, the desired conformation will have a higher binding affinity(and, consequently, a lower dissociation constant) for a cognate ligandof the receptor. For example, the desired conformation of a TNF-bindingmolecule will have a higher binding affinity and a lower dissociationconstant for TNF (e.g., TNF-alpha).

In addition, the desired conformation of a recombinant protein ispreferably more thermostable than an undesired conformation (whenmeasured in the same solution environment). Thermostability can bemeasured in any of a number of ways such as, for example, the meltingtemperature transition (Tm). The desired conformation of a recombinantprotein may or may not have a different arrangement of disulfide bonds,although preferably the conformation contains native disulfide bonds.The desired conformation of a recombinant protein may have othertertiary structure characteristics. For example, a desired conformationmay be a monomer, dimer, trimer, tetramer, or some other higher orderform of the protein. For the purposes of the invention, the“conformation” of a protein is its three-dimensional structure. Twodifferent structures of a polypeptide with the same primary amino acidsequence are “conformers” of each other when they have differentconformations corresponding to energy minima, and they differ from eachother only in the way their atoms are oriented in space. Conformers canbe interconverting (referring to the rotational freedom around bonds tothe exclusion of breaking bonds). Two different structures of apolypeptide with the same primary amino acid sequence are“configurational isomers” when they have different conformationscorresponding to energy minima, they differ from each other in the waytheir atoms are oriented in space, and they are non-interconvertiblewithout the breaking of a covalent bond. In the practice of theinvention, configurational isomers can be interconverted by, forexample, breaking and optionally reforming disulfidc bonds.

Thus, in one aspect, the invention entails contacting a preparation ofthe glycosylatcd recombinant protein that contains a mixture of at leasttwo configurational isomers of the recombinant protein to areduction/oxidation coupling reagent for a time sufficient to increasethe relative proportion of the desired configurational isomer anddetermining the relative proportion of the desired configurationalisomer in the mixture. In another aspect, the invention entailscontacting a preparation of a recombinant protein that has been producedby mammalian cells with a reduction/oxidation coupling reagent, at a pHof about 7 to about 11, and isolating a fraction of the preparation ofthe recombinant protein with a desired conformation. Preferredrecombinant proteins are glycosylated recombinant proteins such as,e.g., those produced by eukaryotic cells.

The invention can be used to treat just about any protein to promote adesired conformation. A protein is generally understood to be apolypeptide of at least about 10 amino acids, more preferably at leastabout 25 amino acids, even more preferably at least about 75 aminoacids, and most preferably at least about 100 amino acids. The methodsof the invention find particular use in treating proteins that have atleast about 3 cysteine residues, more preferably at least about 8cysteine residues, still more preferably at least about 15 cysteineresidues, yet even more preferably at least about 30, still even morepreferably at least about 50 to 150 cysteine residues.

Generally, the methods of the invention are useful for improvingproduction processes for recombinant proteins. Recombinant proteins areproteins produced by the process of genetic engineering. The term“genetic engineering” refers to any recombinant DNA or RNA method usedto create a host cell that expresses a gene at elevated levels, atlowered levels, and/or a mutant form of the gene. In other words, thecell has been transfected, transformed or transduced with a recombinantpolynucleotide molecule, and thereby altered so as to cause the cell toalter expression of a desired protein. Methods and vectors forgenetically engineering cells and/or cell lines to express a protein ofinterest are well known to those skilled in the art; for example,various techniques are illustrated in Current Protocols in MolecularBiology, Ausubel et al., eds. (Wiley & Sons, New York, 1988, andquarterly updates) and Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Laboratory Press, 1989). Genetic engineeringtechniques include but are not limited to expression vectors, targetedhomologous recombination and gene activation (see, for example, U.S.Pat. No. 5,272,071 to Chappel) and trans activation by engineeredtranscription factors (see, for example, Segal el al., 1999, Proc. Natl.Acad. Sci. USA 96(6):2758–63).

The invention finds particular use in improving the production ofproteins that are glycosylated. Specifically, proteins that are secretedby fungal cell systems (e.g., yeast, filamentous fungi) and mammaliancell systems will be glycosylated. Preferably, the proteins are secretedby mammalian production cells adapted to grow in cell culture. Examplesof such cells commonly used in the industry are CHO, VERO, BHK, HeLa,CV1 (including Cos), MDCK, 293, 3T3, myeloma cell lines (especiallymurine), PC12 and WI38 cells. Particularly preferred host cells areChinese hamster ovary (CHO) cells, which are widely used for theproduction of several complex recombinant proteins, e.g. cytokines,clotting factors, and antibodies (Brasel et al., 1996, Blood88:2004–2012; Kaufman et al., 1988, J.Biol Chem 263: 6352–6362; McKinnonet al., 1991, J Mol Endocrinol 6:231–239; Wood et al., 1990, J. Immunol145:3011–3016). The dihydrofolate reductase (DHFR)-deficient mutant cellline (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216–4220), DXB11and DG-44, are the CHO host cell lines of choice because the efficientDHFR selectable and amplifiable gene expression system allows high levelrecombinant protein expression in these cells (Kaufman R. J., 1990, MethEnzymol 185:527–566). In addition, these cells are easy to manipulate asadherent or suspension cultures and exhibit relatively good geneticstability. CHO cells and recombinant proteins expressed in them havebeen extensively characterized and have been approved for use inclinical manufacturing by regulatory agencies.

It has been found that the invention is a gentle and effective processfor improving the production process for proteins that can adoptmultiple conformations and/or contain more than one domain. A “domain”is a contiguous region of the polypeptide chain that adopts a particulartertiary structure and/or has a particular activity that can belocalized in that region of the polypeptide chain. For example, onedomain of a protein can have binding affinity for one ligand, and onedomain of a protein can have binding affinity for another ligand. In athermostable sense, a domain can refer to a cooperative unfolding unitof a protein. Such proteins that contain more than one domain can befound naturally occurring as one protein or genetically engineered as afusion protein. In addition, domains of a polypeptide can havesubdomains.

In one aspect, the methods of the invention can be used on preparationsof recombinant proteins in which at least one domain of the protein hasa stable conformation, and at least one domain of the protein has anunstable conformation. The terms “stable” and “unstable” are used asrelative terms. The domain of the protein with a stable conformationwill have, for example, a higher melting temperature (Tm) than theunstable domain of the protein when measured in the same solution. Adomain is stable compared to another domain when the difference in theTm is at least about 2° C., more preferably about 4° C., still morepreferably about 7° C., yet more preferably about 10° C., even morepreferably about 15° C., still more preferably about 20° C., even stillmore preferably about 25° C., and most preferably about 30° C., whenmeasured in the same solution.

The invention is also generally applicable to proteins that have an Fcdomain, and another domain (e.g., antibodies, and Fc fusion proteins).For example, in one of the non-limiting embodiments illustrated below,TNFR:Fc, the Tm's for the Fc portion of the molecule are at 69.1° C. and83.4° C., while the Tm for the TNFR portion of the molecule range from52.5° C. (in the more desired conformation) to a Tm of 49.7° C. (in theless desired conformation).

Particularly preferred proteins are protein-based drugs, also known asbiologics. Preferably, the proteins are expressed as extracellularproducts. Proteins that can be produced using the methods of theinvention include but are not limited to a flt3 ligand (as described inWO 94/28391, which is incorporated by reference herein in its entirety),a CD40 ligand (as described in U.S. Pat. No. 6,087,329, which isincorporated by reference herein in its entirety), erythropoeitin,thrombopoeitin, calcitonin, Fas ligand, ligand for receptor activator ofNF-kappa B (RANKL), tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL, as described in WO 97/01633, which isincorporated by reference herein in its entirety), thymic stroma-derivedlymphopoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony stimulating factor (GM-CSF, as describedin Australian Patent No. 588819, which is incorporated by referenceherein in its entirety), mast cell growth factor, stem cell growthfactor, epidermal growth factor, RANTES, growth hormone, insulin,insulinotropin, insulin-like growth factors, parathyroid hormone,interferons, nerve growth factors, glucagon, interleukins 1 through 18,colony stimulating factors, lymphotoxin-β, tumor necrosis factor (TNF),leukemia inhibitory factor, oncostatin-M, and various ligands for cellsurface molecules ELK and Hek (such as the ligands for eph-relatedkinases or LERKS). Descriptions of proteins that can be purifiedaccording to the inventive methods may be found in, for example, HumanCytokines: Handbook for Basic and Clinical Research, Vol. II (Aggarwaland Gutterman, eds. Blackwell Sciences, Cambridge, Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook (A. W. Thompson,ed., Academic Press, San Diego, Calif., 1991).

Preparations of the receptors, especially soluble forms of thereceptors, for any of the aforementioned proteins can also be improvedusing the inventive methods, including both forms of TNFR (referred toas p55 and p75), Interleukin-1 receptors types I and II (as described inEP 0 460 846, U.S. Pat. No. 4,968,607, and U.S. Pat. No. 5,767,064,which are incorporated by reference herein in their entirety),Interleukin-2 receptor, Interleukin-4 receptor (as described in EP 0 367566 and U.S. Pat. No. 5,856,296, which are incorporated by referenceherein in their entirety), Interleukin-15 receptor, lnterleukin-17receptor, Interleukin-18 receptor, granulocyte-macrophage colonystimulating factor receptor, granulocyte colony stimulating factorreceptor, receptors for oncostatin-M and leukemia inhibitory factor,receptor activator of NF-kappa B (RANK, as described in U.S. Pat. No.6,271,349, which is incorporated by reference herein in its entirety),receptors for TRAIL (including TRAIL receptors 1, 2, 3, and 4), andreceptors that comprise death domains, such as Fas or Apoptosis-InducingReceptor (AIR).

Other proteins whose production processes can be improved using theinventive methods include cluster of differentiation antigens (referredto as CD proteins), for example, those disclosed in Leukocyte Typing VI(Proceedings of the VIth International Workshop and Conference;Kishimoto, Kikutani et al., eds.; Kobe, Japan, 1996), or CD moleculesdisclosed in subsequent workshops. Examples of such molecules includeCD27, CD30, CD39, CD40; and ligands thereto (CD27 ligand, CD30 ligandand CD40 ligand). Several of these are members of the TNF receptorfamily, which also includes 41BB and OX40; the ligands are often membersof the TNF family (as are 41BB ligand and OX40 ligand); accordingly,members of the TNF and TNFR families can also be produced using thepresent invention.

Proteins that are enzymatically active can also be prepared according tothe instant invention. Examples include metalloproteinase-disintegrinfamily members, various kinases, glucocerebrosidase, superoxidedismutase, tissue plasminogen activator, Factor VIII, Factor IX,apolipoprotein E, apolipoprotein A-I, globins, an IL-2 antagonist,alpha-1 antitrypsin, TNF-alpha Converting Enzyme, and numerous otherenzymes. Ligands for enzymatically active proteins can also be expressedby applying the instant invention.

The inventive compositions and methods are also useful for preparationof other types of recombinant proteins, including immunoglobulinmolecules or portions thereof, and chimeric antibodies (e.g., anantibody having a human constant region coupled to a murine antigenbinding region) or fragments thereof. Numerous techniques are known bywhich DNA encoding immunoglobulin molecules can be manipulated to yieldDNAs capable of encoding recombinant proteins such as single chainantibodies, antibodies with enhanced affinity, or other antibody-basedpolypeptides (see, for example, Larrick et al., 1989, Biotechnology7:934–938; Reichmann et al., 1988, Nature 332:323–327; Roberts et al.,1987, Nature 328:731–734; Verhoeyen et al., 1988, Science 239:1534–1536;Chaudhary et al., 1989, Nature 339:394–397). Preparations of fully humanantibodies (such as are prepared using transgenic animals, andoptionally further modified in vitro), as well as humanized antibodies,can also be used in the invention. The term humanized antibody alsoencompasses single chain antibodies. See, e.g., Cabilly el al., U.S.Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1;Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No.0,120,694 B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S.etal., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No.5,225,539; Winter, European Patent No. 0,239,400 B1; Queen et al.,European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0 519 596A1. The method of the invention may also be used during the preparationof conjugates comprising an antibody and a cytotoxic or luminescentsubstance. Such substances include: maytansine derivatives (such asDM1); enterotoxins (such as a Staphlyococcal enterotoxin); iodineisotopes (such as iodine-125); technium isotopes (such as Tc-99m);cyanine fluorochromes (such as Cy5.5.18); and ribosome-inactivatingproteins (such as bouganin, gelonin, or saporin-S6).

Examples of antibodies or antibody/cytotoxin or antibody/luminophoreconjugates contemplated by the invention include those that recognizeany one or combination of the above-described proteins and/or thefollowing antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22,CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147,IL-1α, IL-1β, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor,IL-6 receptor, IL-13 receptor, IL-18 receptor subunits, PDGF-β, VEGF,TGF, TGF-β2, TGF-β1, EGF receptor, VEGF receptor, C5 complement, IgE,tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which is agene product that is expressed in association with lung cancer), HER-2,a tumor-associated glycoprotein TAG-72, the SK-1 antigen,tumor-associated epitopes that are present in elevated levels in thesera of patients with colon and/or pancreatic cancer, cancer-associatedepitopes or proteins expressed on breast, colon, squamous cell,prostate, pancreatic, lung, and/or kidney cancer cells and/or onmelanoma, glioma, or neuroblastoma cells, the necrotic core of a tumor,integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins, TRAILreceptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, the adhesionmolecule VAP-1, epithelial cell adhesion molecule (EpCAM), intercellularadhesion molecule-3 (ICAM-3), leukointegrin adhesin, the plateletglycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroidhormone, rNAPc2 (which is an inhibitor of factor VIIa-tissue factor),MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumornecrosis factor (TNF), CTLA-4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, L-selectin, IFN-γ, Respiratory Syncitial Virus, humanimillimolarunodeficiency virus (HIV), hepatitis B virus (HBV),Streptococcus mutans, and Staphlycoccus aureus.

Preparations of various fusion proteins can also be prepared using theinventive methods. Examples of such fusion proteins include proteinsexpressed as a fusion with a portion of an immunoglobulin molecule,proteins expressed as fusion proteins with a zipper moiety, and novelpolyfunctional proteins such as a fusion proteins of a cytokine and agrowth factor (i.e., GM-CSF and IL-3, MGF and IL-3). WO 93/08207 and WO96/40918 describe the preparation of various soluble oligomeric forms ofa molecule referred to as CD40L, including an immunoglobulin fusionprotein and a zipper fusion protein, respectively; the techniquesdiscussed therein are applicable to other proteins. Any of the abovemolecules can be expressed as a fusion protein including but not limitedto the extracellular domain of a cellular receptor molecule, an enzyme,a hormone, a cytokine, a portion of an immunoglobulin molecule, a zipperdomain, and an epitope.

The preparation of recombinant protein can be a cell culturesupernatant, cell extract, but is preferably a partially purifiedfraction from the same. By “partially purified” means that somefractionation procedure, or procedures, have been carried out, but thatmore polypeptide species (at least 10%) than the desired protein orprotein conformation is present. One of the advantages of the methods ofthe invention is that the preparation of recombinant protein can be at afairly high concentration. Preferred concentration ranges are 0.1 to 20mg/ml, more preferably from 0.5 to 15 mg/ml, and still more preferablyfrom 1 to 10 mg/ml.

The preparation of recombinant protein can be prepared initially byculturing recombinant host cells under culture conditions suitable toexpress the polypeptide. The polypeptide can also be expressed as aproduct of transgenic animals, e.g., as a component of the milk oftransgenic cows, goats, pigs, or sheep which are characterized bysomatic or germ cells containing a nucleotide sequence encoding thepolypeptide. The resulting expressed polypeptide can then be purified,or partially purified, from such culture or component (e.g., fromculture medium or cell extracts or bodily fluid) using known processes.Fractionation procedures can include but are not limited to one or moresteps of filtration, centrifugation, precipitation, phase separation,affinity purification, gel filtration, ion exchange chromatography,hydrophobic interaction chromatography (HIC; using such resins as phenylether, butyl ether, or propyl ether), HPLC, or some combination ofabove.

For example, the purification of the polypeptide can include an affinitycolumn containing agents which will bind to the polypeptide; one or morecolumn steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving elution; and/or immunoaffinity chromatography. The polypeptidecan be expressed in a form that facilitates purification. For example,it may be expressed as a fusion polypeptide, such as those of maltosebinding polypeptide (MBP), glutathione-S-transferase (GST) orthioredoxin (TRX). Kits for expression and purification of such fusionpolypeptides are commercially available from New England BioLab(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogcn,respectively. The polypeptide can be tagged with an epitope andsubsequently purified by using a specific antibody directed to suchepitope. One such epitope (FLAG®) is commercially available from Kodak(New Haven, Conn.). It is also possible to utilize an affinity columncomprising a polypeptide-binding polypeptide, such as a monoclonalantibody to the recombinant protein, to affinity-purify expressedpolypeptides. Other types of affinity purification steps can be aProtein A or a Protein G column, which affinity agents bind to proteinsthat contain Fc domains. Polypeptides can be removed from an affinitycolumn using conventional techniques, e.g., in a high salt elutionbuffer and then dialyzed into a lower salt buffer for use or by changingpH or other components depending on the affinity matrix utilized, or canbe competitively removed using the naturally occurring substrate of theaffinity moiety. In one embodiment of the invention illustrated below,the preparation of recombinant protein has been partially purified overa Protein A affinity column.

Some or all of the foregoing purification steps, in variouscombinations, can also be employed to prepare an appropriate preparationof a recombinant protein for use in the methods of the invention, and/orto further purify the recombinant polypeptide after contacting thepreparation of the recombinant protein with a reduction/oxidationcoupling reagent. The polypeptide that is substantially free of othermammalian polypeptides is defined as an “isolated polypeptide”.

The polypeptide can also be produced by known conventional chemicalsynthesis. Methods for constructing polypeptides by synthetic means areknown to those skilled in the art. The synthetically-constructedpolypeptide sequences can be glycosylated in vitro.

The desired degree of final purity depends on the intended use of thepolypeptide. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no polypeptide bandscorresponding to other polypeptides are detectable upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto the polypeptide can be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.Most preferably, the polypeptide of the invention is purified tosubstantial homogeneity, as indicated by a single polypeptide band uponanalysis by SDS-PAGE. The polypeptide band can be visualized by silverstaining, Coomassie blue staining, and/or (if the polypeptide isradiolabeled) by autoradiography.

By “contacting” is meant subjecting to, and/or exposing to, in solution.The protein or polypeptide can be contacted while also bound to a solidsupport (e.g., an affinity column or a chromatography matrix).Preferably, the solution is buffered. In order to maximize the yield ofprotein with a desired conformation, the pH of the solution is chosen toprotect the stability of the protein and to be optimal for disulfideexchange. In the practice of the invention, the pH of the solution ispreferably not strongly acidic. Thus, preferred pH ranges are greaterthan pH 5, preferably about pH 6 to about pH 11, more preferably fromabout pH 7 to about pH 10, and still more preferably from about pH 7.6to about pH 9.6. In one non-limiting embodiment of the invention usingTNFR:Fc that is illustrated below, the optimal pH was found to be aboutpH 8.6. However, the optimal pH for a particular embodiment of theinvention can be easily determined experimentally by those skilled inthe art.

The reduction/oxidation coupling reagent is a source of reducing agents.Preferred reducing agents are free thiols. The reduction/oxidationcoupling reagent is preferably comprised of a compound from the groupconsisting of reduced and oxidized glutathione, dithiothreitol (DTT),2-mercaptoethanol, dithionitrobenzoate, cysteine and cystine. For easeof use and economy, reduced glutathione and/or reduced cysteine can beused.

The reduction/oxidation coupling reagent is present at a concentrationsufficient to increase the relative proportion of the desiredconformation. The optimal concentration of the reduction/oxidationcoupling reagent depends upon the concentration of protein and number ofdisulfide bonds in the protein. For example, it has been found using aprotein (TNFR:Fc) with 29 disulfide bonds at a concentration of 2 mg/ml(approximately 14 microM protein or 400 microM disulfide), areduction/oxidation coupling reagent with 2 mM reduced thiols workedwell to increase the relative proportion of the desired conformation.This corresponds to a ratio of about 35 free thiols to 1 disulfide bond.However, it was also found that ratios from 20 to 400 free thiols perdisulfide also worked. Of course, the amount of thiol used for aparticular concentration can vary somewhat depending upon the reducingcapacity of the thiol, and can be easily determined by one of skill inthe art.

Thus, generally, the concentration of free thiols from thereduction/oxidation coupling reagent can be from about 0.05 mM to about50 mM, more preferably about 0.1 mM to about 25 mM, and still morepreferably about 0.2 mM to about 20 mM.

In addition, the reduction/oxidation coupling reagent can containoxidized thiols at approximately higher, equal or lower concentrationsas the reduced thiol component. For example, the reduction/oxidationcoupling reagent can be a combination of reduced glutathione andoxidized glutathione. It has been found through actual working examples,that a ratio of reduced glutathione to oxidized glutathione of fromabout 1:1 to about 100:1 (reduced thiols:oxidized thiols) can functionequally well. Alternatively in another embodiment, thereduction/oxidation coupling reagent can be cysteine or a combination ofcysteine and cystine. Thus, when oxidized thiols are included in theinitial reduction/oxidation coupling reagent, the ratio of reducedthiols to oxidized thiols can in a preferred embodiment be from about1:10 to about 1000:1, more preferably about 1:1 to about 500:1, stillmore preferably about 5:1 to about 100:1, even more preferably about10:1.

Contacting the preparation of recombinant protein with areduction/oxidation coupling reagent is performed for a time sufficientto increase the relative proportion of the desired conformation. Anyrelative increase in proportion is desirable, but preferably at least10% of the protein with an undesired conformation is converted toprotein with the desired conformation. More preferably at least 20%,30%, 40%, 50%, 60%, 70% and even 80% of the protein is converted from anundesired to a desired conformation. Typical yields that have beenachieved with the methods of the invention range from 40 to 80%. If thecontacting step is performed on a partially or highly purifiedpreparation of recombinant protein, the contacting step can be performedfor as short as about 1 hour to about 4 hours, and as long as about 6hours to about 4 days. It has been found that a contacting step of about4 to about 16 hours or about 18 hours works well. The contacting stepcan also take place during another step, such as on a solid phase orduring filtering or any other step in purification.

The methods of the invention can be performed over a wide temperaturerange. For example, the methods of the invention have been successfullycarried out at temperatures from about 4° C. to about 37° C., howeverthe best results were achieved at lower temperatures. A typicaltemperature for contacting a partially or fully purified preparation ofthe recombinant protein is about 4° C. to about 25° C. (ambient), butcan also be performed at lower temperatures and at higher temperature.

The preparation of recombinant protein can be contacted with thereduction/oxidation coupling reagent in various volumes as appropriate.For example, the methods of the invention have been carried outsuccessfully at the analytical laboratory-scale (1–50 mL),preparative-scale (50 mL–10 L) and manufacturing-scale (10 L or more).Thus, the methods of the invention can be carried out on both small andlarge scale with reproducibility.

In preferred aspects, the contacting step is performed in the absence ofsignificant amounts of chaotropic agents such as, for example, SDS, ureaand guanidium HCl. Significant amounts of chaotropic agents are neededto observe perceptible unfolding. Generally, less than 1 M chaotrope ispresent, more preferably less than 0.5 M, still more preferably lessthan 0.1 M chaotrope. A solution is essentially free of chaotrope (e.g.,SDS, urea and guanidium HCl) when no chaotrope has been purposely addedto the solution, and only trace levels (e.g., less than 10 mM) may bepresent (e.g., from the vessel or as a cellular byproduct).

Disulfide exchange can be quenched in any way known to those of skill inthe art. For example, the reduction/oxidation coupling reagent can beremoved or its concentration reduced through a purification step, and/orit can be chemically inactivated by, e.g., acidifying the solution.Typically, when the reaction is quenched by acidification, the pH of thesolution containing the reduction/oxidation coupling reagent will bebrought down below pH 7. Preferably, the pH is brought to below pH 6.Generally, the pH is reduced to between about pH 2 and about pH 7.

Determining the conformation of a protein, and the relative proportionsof a conformation of a protein in a mixture, can be done using any of avariety of analytical and/or qualitative techniques. If there is adifference in activity between the conformations of the protein,determining the relative proportion of a conformation in the mixture canbe done by way of an activity assay (e.g., binding to a ligand,enzymatic activity, biological activity, etc.). For example, in one ofthe non-limiting embodiments described below, at least two differentconformations of TNFR:Fc can be resolved by using a solid-phase TNFbinding assay. The assay, essentially as described for IL-1R (Slack, etal., 1993, J. Biol. Chem. 268:2513–2524), can differentiate between therelative proportions of various protein conformations by changes inligand-receptor binding association, dissociation or inhibitionconstants generated. Alternatively the binding results can be expressedas activity units/mg of protein.

If the two conformations resolve differently during chromatography,electrophoresis, filtering or other purification technique, then therelative proportion of a conformation in the mixture can be determinedusing such purification techniques. For example, in the non-limitingembodiments described below, at least two different conformations ofTNFR:Fc could be resolved by way of hydrophobic interactionchromatography. Further, since far-UV Circular Dichroism has been usedto estimate secondary structure composition of proteins (Perczel et al.,1991, Protein Engrg. 4:669–679), such a technique can determine whetheralternative conformations of a protein are present. Still anothertechnique used to determine conformation is fluorescence spectroscopywhich can be employed to ascertain complimentary differences in tertiarystructure assignable to tryptophan and tyrosine fluorescence. Othertechniques that can be used to determine differences in conformationand, hence, the relative proportions of a conformation, are on-line SECto measure aggregation status, differential scanning calorimetry tomeasure melting transitions (Tm's) and component enthalpies, andchaotrope unfolding.

By the term “isolating” is meant physical separation of at least onecomponent in a mixture away from other components in a mixture.Isolating components or particular conformations of a protein can beachieved using any purification method that tends to separate suchcomponents. Accordingly, one can perform one or more chromatographysteps, including but not limited to HIC, hydroxyapatite chromatography,ion exchange chromatography, affinity, and SEC. Other purificationmethods are filtration (e.g., tangential flow filtration),electrophoretic techniques (e.g., electrophoresis, electroelution,isoelectric focusing), and phase separation (e.g., PEG-dextran phaseseparation), to name just a few. In addition, the fraction of thepreparation of recombinant protein that contains the protein in theundesired conformation can be treated again in the methods of theinvention, to further optimize the yields of protein with the desiredconformation.

For example, after treatment, protein samples can be prepared forhydrophobic interaction chromatography (HIC) by the following method. Anequal volume of 850 mM sodium citrate, 50 mM sodium phosphate, pH 6.5 isadded to the treated sample, and allowed to equilibrate to roomtemperature. After filtering (e.g., using a 0.22 □m filter), HICchromatography is performed on a Toyopearl® Butyl 650-M resin (TosohBiosep LLC, Montgomeryville, Pa.), at a flow rate of 150 cm/hr, and amass load of 2.1 mg•mL resin⁻¹. The column is prequilibrated with 3column volumes of 425 mM NaCitrate, 50 mM PO₄ pH 6.5, sample is loaded,and then washed through with 3 column volumes of 425 mM NaCitrate, 50 mMPO₄ pH 6.5. Elution can be performed with a gradient of 425 mMNaCitrate, 50 mM PO₄ pH 6.5 to O mM NaCitrate, 50 mM PO₄ pH 6.5 in atotal of 5 column volumes. Fractions can be collected during theelution. The column can be stripped with 3 column volumes of waterfollowed by 3 column volumes of 0.1M NaOH. Using the methods of theinvention accordingly, one can thus obtain preparations of TNFR:Fc thatcontain more than 85%, more than 90%, and even more than 95% of theTNFR:Fc present in the preparation in the most active conformation(Fraction #2). Compositions, including pharmaceutical compositions, ofTNFR:Fc containing such proportions of Fraction #2 are therefore alsoprovided by the invention.

The invention also optionally encompasses further formulating theproteins. By the term “formulating” is meant that the proteins can bebuffer exchanged, sterilized, bulk-packaged and/or packaged for a finaluser. For purposes of the invention, the term “sterile bulk form” meansthat a formulation is free, or essentially free, of microbialcontamination (to such an extent as is acceptable for food and/or drugpurposes), and is of defined composition and concentration. The term“sterile unit dose form” means a form that is appropriate for thecustomer and/or patient administration or consumption. Such compositionscan comprise an effective amount of the protein, in combination withother components such as a physiologically acceptable diluent, carrier,and/or excipient. The term “pharmaceutically acceptable” means anon-toxic material that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). Formulations suitablefor administration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. In addition, sterilebulk forms and sterile unit forms may contain a small concentration(approximately 1 microM to approximately 10 mM) of a reduction/oxidationcoupling reagent (e.g., glutathione, cysteine, etc.). The polypeptidescan be formulated according to known methods used to preparepharmaceutically useful compositions. They can be combined in admixture,either as the sole active material or with other known active materialssuitable for a given indication, with pharmaceutically acceptablediluents (e.g., saline, Tris-HCl, acetate, and phosphate bufferedsolutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens),emulsifiers, solubilizers, adjuvants and/or carriers. Suitableformulations for pharmaceutical compositions include those described inRemington's Pharmaceutical Sciences, 16th ed. 1980, Mack PublishingCompany, Easton, Pa. In addition, such compositions can be complexedwith polyethylene glycol (PEG), metal ions, and/or incorporated intopolymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. No. 4,235,871;U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No.4,737,323. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance, and are thus chosen according to the intended application, sothat the characteristics of the carrier will depend on the selectedroute of administration. Sustained-release forms suitable for useinclude, but are not limited to, polypeptides that are encapsulated in aslowly-dissolving biocompatible polymer (such as the alginatemicroparticles described in U.S. Pat. No. 6,036,978), admixed with sucha polymer (including topically applied hydrogels), and or encased in abiocompatible semi-permeable implant.

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

EXAMPLE 1 Biophysical Assessment of TNFR:Fc Fractions #2 and #3

The p75 TNFR:Fc elutes off a hydrophobic interaction column (HIC) asthree distinct peaks termed Fraction #1, Fraction #2 and Fraction #3(see FIG. 1). Fraction #2 is the desired fraction. Fraction #3 was ofparticular interest since it can comprise from 20 to 60% of the sampleand has been shown to exhibit low TNF binding activity and A375bioactivity in comparison with Fraction #2. Therefore, in the interestof understanding the differences between these two fractions andascertaining what factors contribute to the loss in activity forFraction #3 as it pertains to structure and conformation, biophysicalstudies were carried out. In this example, we analyzed Fraction #2 andFraction #3 using Circular Dichroism, Fluorescence, on-lineSEC/UV/LS/RI, and differential scanning calorimetry (DSC).

Materials and Methods

Materials: The starting material was TNFR:Fc in TMS buffer (10 mM Tris,4% mannitol, 1% sucrose). HIC eluted fractions of this material wereisolated as Fractions #2 and #3 for experimental studies describedbelow.

Circular Dichroisim: Studies were carried out in the near (250–340 nm)and far-UV (190–250 nm) regions. The near-UV studies were carried out toelucidate differences in tertiary structure while the far-UV studieswere used to characterize differences in secondary structure.

The near-UV Circular Dichroism measurements were conducted in the TMSsolutions with the following concentrations. Starting material wasdiluted to 6.25 mg/ml while the Fractions #2 and #3 were evaluated attheir existing concentrations of 9.4 and 5.4 mg/ml, respectively. ACircular Dichroism cell with a path length of 0.1 cm was used and scansconducted from 340 to 250 nm.

The far-UV Circular Dichroism measurements were performed with theprotein buffer exchanged into 10 mM sodium phosphate (pH 7.0) andsubsequently evaluated using a 0.1 cm path length cell scanned from 250to 190 nm. Secondary structure composition was evaluated using convexconstraint analysis (CCA) (Perczel et al., 1991, Protein Engrg.4:669–679).

Fluorescence Spectroscopy: Samples were examined after dilution toapproximately 50 microgram/ml using two different excitationwavelengths. Tyrosine and tryptophan fluorescence was examined with anexcitation of 270 nm while tryptophan fluorescence was exclusivelyevaluated using an excitation of 295 nm (Lakowicz, J. R. in “Principlesof Fluorescence Spectroscopy”, Plenum Press, 1983. New York, N.Y.,342–343). Fluorescence scans extended from 300 to 440 nm for 270 nmexcitation and from 310 to 440 nm for 295 nm excitation. Fourconsecutive scans were signal averaged for each spectrum. Normalizeddata were reported to evaluate differences in frequency arising from thesamples.

Online-SEC/UV/LS/RI: The molecular weights of eluting components usingsize exclusion chromatography were ascertained using ultraviolet (UV @280 nm), light scattering (90°), and refractive index (RI) detection inseries. This method has been well documented (see Arakawa et al., 1992,Anal. Biochem. 203:53–57 and Wen et al., 1996, Anal. Biochem.240:155–166), and has an advantage of measuring the nonglycosylatedmolecular weights of proteins and peptides that are glycosylated. TheSEC and UV data were collected using an Integral HPLC system (PerSeptiveBiosystems, Inc.) with a BioSil-400-5 column (from BioRad) using a flowrate of 1 ml/min. The elution buffer consisted of 100 mM phosphate (pH6.8) and 100 mM NaCl. A DAWN DSP multi-angle light scattering detectorand Optilab DSP refractometer were both purchased from Wyatt Technology,Inc. Calibration standards to determine instrumental constants includedBSA dimer, BSA monomer and ovalbumin (FIG. 2).

Differential Scanning Calorimetry (DSC): Physical properties ofunfolding were measured using a MicroCal MC-2 DSC instrument in upscanmode. Samples were prepared by buffer exchanging into the same TMSbuffer at pH 7.4. Samples contained about 4 mg/ml protein and wereevaluated against the buffer (absent protein) as a reference. The scanrate was 67° C./hr spanning the temperature regime from 20° C. to 90° C.Collected scans were subsequently converted into concentrationnormalized scans to better compare enthalpic behavior of unfoldingtransitions while taking into account differences in concentration (datareported as kcal/mole).

Results

Circular Dicliroism. The near-UV Circular Dichroism measurementsexpressed in terms of mean residue ellipticity are shown in FIG. 2.Changes in a broad feature near 270 nm were evident between Fraction #2and #3 as shown by a greater proportion of negative ellipticity in thespectrum of Fraction #3 (indicated by the arrow in FIG. 2A). It wasnoted that the spectral behavior of the starting material closelymatches that of Fraction #2 but does exhibit a subtle negativedisplacement in the same region surrounding 270 nm. This result seemedconsistent as Fraction #3 made up a small part of the starting materialand so its contribution to the overall ellipticity in this region wasgreatly reduced but in the same displacement direction. Reproducibilityof the Fraction #3 spectrum confirmed the observed displacement of thissample to be real. With this in mind, and knowing that disulfides giverise to a broad negative elliptical feature in this region of theCircular Dichroism spectrum (see Kahn, P. C., 1978, Methods Enzymol.61:339–378 and Kosen et al., 1981, Biochemistry 20:5744–5754), thenear-UV Circular Dichroism spectrum was curve-fitted to estimate whatthe observed changes in this region mean in terms of tertiary structure.The results of the curve-fitted data are presented in FIG. 2B and showeda small red-shift (3 nm) and enhanced negative displacement consistentwith the contribution arising from a change in tertiary structureinvolving disulfides when comparing Fraction #3 with #2.

The far-UV Circular Dichroism has been used to estimate secondarystructure composition of proteins (Perczel et al., 1991, Protein Engrg.4:669–679). Secondary structure assignments using CCA were performed.Calculated spectra comprised of the sum of the secondary structureelements were compared with experimentally observed spectra andexhibited a good fit. The secondary structures of both fractions werecomparable within limits of experimental precision (within 10%).Therefore, this experiment did not distinguish any differences regardingsecondary structure for either of these two fractions.

Fluorescence Spectroscopy. Knowing that there were significantdifferences observed in the near-UV Circular Dichroism region,fluorescence spectroscopy was employed to ascertain complimentarydifferences in tertiary structure assignable to tryptophan and tyrosinefluorescence. Using two excitation wavelengths, it was possible todetermine that the spectra for all three cases considered (SM, Fraction#2 and #3) were super-imposable with fluorescence maxima near 338 nm.Since the three-dimensional structure of a given protein is responsiblefor emission maxima of native proteins, these results suggested that theaverage structure involving the intrinsic fluorophores, tryptophan andtyrosine was unperturbed.

On-line SEC/UV/LS/RI. The light scattering studies performed on-linewith SEC yielded molecular weights of the main elution peak that were inagreement with the non-glycosylated polypeptide molecular weight ofTNFR:Fc (e.g., 102 kD). Although there were clear differences in thecompositions of eluting species evaluated with this technique, whencomparing the elution profile of Fraction #3 with Fraction #2 (FIG. 3Aand B), the main peak comprising the majority component was measured tobe 102.5±1.6 kD (Retention Volume=8.4 mL) and 101.9±2.1 kD (RetentionVolume=8.3 mL), respectively. The precision was expressed as thestandard deviation of 23 slices through the elution peak bracketed bythe vertical dashed lines in FIG. 3. It was also noted that arespectable signal of the descending shoulder for Fraction #3 permitteddetermination of the polypeptide molecular weight to be 78.1±3.7 kD(this evaluation considered 8 slices surrounding the peak labeled at8.85 mL). As exhibited by the precision associated with the molecularweight determination of this component, this peak exhibited greaterheterogeneity and as a result was suspect of greater polydispersion thanthe main peak. Fraction #3 also contained a significant amount of highmolecular weight species consistent with the elution volume of apredominantly dimeric form of TNFR:Fc (near 7.5). Hence, it wasdetermined that Fraction #3 is comprised of several species includingaggregates and fragmented portions of the molecule.

Differential Scanning Calorimetry. DSC measurements carried out on thetwo fractions yielded significant differences in the unfolding of theTNFR moiety of the TNFR:Fc molecule (FIG. 4). As shown more clearly inthe baseline corrected data (FIG. 4B), there is a 2.8° C. shift to lowertemperature in the melting transition (Tm) when comparing a Tm of 52.5°C. (Fraction #2) with a Tm of 49.7° C. (Fraction #3). The transition isslightly broader for Fraction #3 with a half-width at half thetransition maximum of 8° C. in comparison with Fraction #2 having ahalf-width of 6.5° C. This low temperature transition has beenidentified from thermal unfolding experiments of TNFR:Fc monomer to bedue the TNFR domain of the molecule. Thermal transitions at 69.1° C. and83.4° C. have been assigned to the Fc portion of the molecule. Theselatter two unfolding transitions align well and are comparable in termsof Tm's and component enthalpies.

Discussion

Among the methods tested, differences were observed in the near-UVCircular Dichroism and DSC measurements. Differential scanningcalorimetry data support a loosening of structure that is assignable tothe receptor moiety of the molecule with little change observed in theregion of the Fc. The near-UV Circular Dichroism results suggested thatdisulfides are involved with tertiary structural changes associated withFraction #3. These changes may arise as a consequence of burieddisulfides gaining more exposure to the solvent and account for anincrease in hydrophobicity as suggested by the small increase inretention time observed in the HIC elution of Fraction #3. It isinteresting that there are no discernible differences found in thefluorescence data that would indicate such a change in conformationalstructure. If one considers the primary structure of TNFR:Fc in terms ofthe distribution of tyrosines (Y) and tryptophans (W), it becomesapparent that the region extending from the C-terminal portion ofresidue 104 of the TNFR domain to residue 296 of the N-terminal portionof the Fc (comprising 40% of the linear sequence of TNFR:Fc) is devoidof these intrinsic fluorophores. Therefore, one possible explanationconsistent with the data might be that tertiary structure remote fromthe Fc hinge region is relatively unchanged while that from aboutresidue C115 to C281 may be somewhat altered conformationally. Thisregion of the molecule comprises 10 possible cysteines that may beaffected with supposedly little consequence of structural changeaffecting local structure of tyrosines and tryptophans. It is noted thatit is currently unknown as to how this molecule is folded and it wouldseem plausible that the cysteines that make up disulfides that are moreremote from any given tryptophan or tyrosine residue would be logicalsuspects for tertiary structural changes that produce the observednear-UV Circular Dichroism results but exhibit little impact on thevicinal structure involving tyrosines and tryptophans. This idea doesnot preclude the possibility that there is some unusual change instructure within one or both of the TNFR arms that does not invoke anappreciable change in the net effect of fluorescence arising fromtyrosines and tryptophans. The fact that the fluorescence data (which isinsensitive to disulfides) show no change and the near-UV (that issensitive to disulfides, tyrosines, and tryptophans) exhibits a smallnegative displacement consistent with disulfide structural modificationdoes imply that disulfides play a role in the difference betweenFractions #2 and #3.

In summarizing the remaining data generated concerning Fraction #3,aspects related to molecular weight and secondary structure were foundto be indistinguishable from Fraction #2.

EXAMPLE 2 Disulfide Exchange Experiments on TNFR:Fc Fraction #3 withGlutathione

This experiment was designed to assess a variety of treatments to driveTNRF:Fc Fraction #3 into the conformation of Fraction #2 in a processamenable to large-scale production runs.

Materials and Methods

Materials. The starting material was TNFR:Fc as a Protein A elute, apure HIC elute of Fraction #3, and a 50:50 mixture of HIC elutesFraction #2 and Fraction #3. Buffers were 0.1 M citrate or 0.1 MTris/glycine at pH 7.6, pH 8.6 or pH 9.6. Protein concentration of theTNFR:Fc was from 0.2 to 4.5 mg/mL. A redox coupling system of reducedglutathione and glutathione (GSH/GSSG at a ratio of 10:1) was added at0.1 to 5 mM GSH. Incubation temperature was varied at 4 degrees, 22degrees or 31 degrees Centigrade.

Methods. Disulfide exchange was quenched by acidification of the sampleto pH 6 with 1 M acetic acid. Treated preparations of recombinantprotein were characterized by analytical HIC, SEC (retention time,aggregate concentration) and solid-phase TNF binding assay to determinethe percentage and yield of Fraction #2.

Results and Discussion

Treatment efficiency as a function of pH and GSH concentration.Significant % of the protein in Fraction #3 (at least 10%) was convertedFraction #2 when treatment was performed at both 0.1 mM GSH/pH 7.6 and0.1 mM GSH/pH 8.6. However, efficiency was greatly improved (from 45% toalmost 70%) when treatment was performed at 0.1 mM GSH/pH 9.6; 1 mMGSH/pH 7.6; 1 mM GSH/pH 8.6; and 1 mM GSH/pH 9.6. Thus, althoughtreatment efficiency is sensitive to pH and free thiol concentration, itcan be effectively performed over a wide range of these variables.

Temperature effects. Fraction #3 was treated at three differenttemperatures, 4° C., 22° C. and 31° C. The GSH concentration was held at1 mM, and pH 8.6. After 16 hours, the treatment groups all exhibitedsignificant conversion of Fraction #3 into Fraction #2, but conversionseemed slightly more efficient at the two lower temperatures.

Clone effects. Six different cell line clones, all producing TNFR:Fc,were tested in a standardized protocol based upon the above results.Specifically, a Protein A elution containing 0.4 to 0.7 mg/mL of TNFR:Fc(at about pH 4) was adjusted to pH 8.6 using 1M Tris/glycine (finalconcentration 0.1 M Tris/glycine). These solutions were adjusted to 1 mMEDTA and 2.5 mM GSH/0.25 mM GSSG and incubated at room temperature forabout 16 hours. Disulfide exchange was quenched by acidification asdescribed above.

Each of six different clones all showed improvement in production andyield of Fraction #2. The reduction of HIC Fraction #3 by treatment inthe various clones was 64%, 72%, 77%, 78%, 78% and 83%. The increase inHIC Fraction #2 in the same clones was 37%, 64%, 78%, 70%, 44% and 54%,respectively. Percent increase in HIC Fraction #2 was well correlatedwith the % increase in Binding Units, as shown in FIG. 5. Thus, themethods appeared generally applicable across all clones tested.

Binding assays. Three different preparations of TNFR:Fc were assayed ina solid-phase TNF binding assay. Samples 11-6 and 12 were eluants from aProtein A column. Sample 8085-47 was also eluted from a Protein Acolumn, and then subjected to an additional HIC purification step; thissample contained exclusively Fraction #3. Samples were examined in thebinding assay before and after disulfide exchange as described above.The results presented below in Table 1 show an increase in ligandbinding activity after treatment of all samples with glutathione.

TABLE 1 TNF binding activity of TNFR:Fc before and after disulfideexchange Pre-exchange Post-exchange % Sample (activity/mg of protein)Change 11-6 4.16 × 10⁷ 5.73 × 10⁷ 27% 12 4.36 × 10⁷ 6.13 × 10⁷ 29%8085-47 1.90 × 10⁷ 6.75 × 10⁷ 72%

EXAMPLE 3 Disulfide Exchange Experiments on TNFR:Fc Treated withL-Cysteine

This experiment was designed to assess cysteine/cystine asreduction/oxidation coupling reagents for TNFR:Fc. The procedure allowsassessment of change of HIC Fraction #3 into the conformation ofFraction #2 in a process amenable to large-scale production runs. Theprocedure can be performed on a purified Fraction #3, a mixture ofFractions #2 and #3, and/or following other separation techniques suchas Protein A chromatography, with similar results.

Materials and Methods

The starting material was TNFR:Fc as a pure HIC elute of Fraction #3 oras a Protein A-eluted TNFR:Fc containing both Fraction #2 and #3.Buffers were 0.1 M citrate or 0.2 M Tris at pH 8.5. Proteinconcentration of the TNFR:Fc was 2.5 to 3 mg/mL.

A redox coupling system of L-cysteine (varying from 0 to 50 mM) wasutilized. The procedure was also assessed +/−L-cystine (0.025 to 0.5 mM)and +/−1 mM EDTA. Incubation temperature was assessed at 4, 15, and 22degrees Centigrade for 6, 18, and 48 hours. Disulfide exchange wasquenched by acidification of the sample to pH 7 with NaH₂PO₄ or 0.85 Mcitrate. Treated preparations of recombinant protein were characterizedby analytical HIC and SEC (retention time, aggregate concentration) todetermine the percentage and yield of Fraction #2 and Fraction #3,cysteinylation and free sulfhydral assays.

Results and Discussion

Treatment efficiency as a function of L-cysteine concentration (0–5 mM).A significant percentage of the TNFR:Fc protein in HIC Fraction #3(average 10%) was converted to Fraction #2 when treatment was performedwith 0.25 mM L-cysteine in the absence of L-cystine or EDTA in fourreplicate samples (FIG. 6). However, efficiency was greatly improved(from 45% to almost 70%) when treatment was performed at 1 mM L-cysteineor 5 mM L-cysteine (FIG. 6). The effect of cystine in these reactionconditions varied with EDTA presence (see below). For a given cellculture batch (samples from four different cell culture batches weretreated), the treatment process was reproducible.

Treatment efficiency as a function of higher L-cysteine concentration(5–50 mM). Higher concentrations of L-cysteine (5, 15 and 50 mML-cysteine) used to treat TNFR:Fc resulted in a decrease in HIC Fraction#3 from the starting material in each case, but 5 mM L-cysteine was mosteffective in promoting the accumulation of Fraction #2 (FIG. 7). It isestimated that higher concentrations of L-cysteine either significantlyreduced the sulfhydryl moieties in the molecule or required too long tore-oxidize.

Treatment efficiency as a function of additional L-cysteine feeding. Inorder to attempt to increase disulfide exchange efficiency, TNFR:Fc wastreated with 5 mM L-cysteine and incubated at 4 degrees Centigrade for18 hours. Additional L-cysteine (0–5 mM) was then added, and the samplesincubated at 4 degrees Centigrade for two additional days. Under theseconditions, no significant effect on the ratio of HIC Fraction #3 toFraction #2 was noted by additional L-cysteine feeding.

Effect of EDTA, cystine and L-cysteine. The effect of cystine (0–0.4mM)in combination with L-cystcine (5 mM) on TNFR:Fc was assessed in thepresence or absence of 1 mM EDTA. Optimal results in the presence of 1mM EDTA occurred with concentrations of cystine in the range of 0.1–0.2mM.

Copper, temperature and time effects. TNFR:Fc was treated at with 5 mML-cysteine at 4 degrees and 22 degrees Centigrade for either 6 or 18hours. Completion of treatment of TNFR:Fc was assayed by copper additionfollowed by HIC. After 6 hours of incubation, disulfide exchange is morecomplete at 4 degrees than 22 degrees, and treatment is clearly morecomplete after 18 hours at 4 degrees Centigrade (FIGS. 8A and 8B).

Comparison of analytical- versus preparative-scale L-cysteine treatmentefficiency. Based upon the treatment conditions optimized at smallscale, TNFR:Fc (2.5 mg/mL in 0.2 M Tris, pH 8.5) in either 3 mL or 20 Lquantities were treated with 5 mM L-cysteine (in the absence of cystineor EDTA), incubated at 4 degrees Centigrade for 18 hours, diluted withand equal volume of 850 mM sodium citrate, 50 mM sodium phosphate, pH6.5 to quench the treatment, and chromatographed on HIC. Control samplesof Preparative and Analytical scale TNFR:Fc had 63% and 68% Fraction #3,respectively. After treatment with the above conditions, Fraction #3 wasreduced to 28% in both Preparative and Analytical scales. Therefore thetreatment efficiency was 56% and 59% for the Preparative and Analyticalsamples, respectively (Table 2). This experiment demonstrates that theprocess is amenable to larger scale treatment.

TABLE 2 Analytical vs. Preparative Scale Disulfide Exchange ProcedurePREPARATIVE ANALYTICAL Fraction #2 Fraction #3 Fraction #2 Fraction #3Control 37% 63% 32% 68% Exchange 72% 28% 72% 28% Exchange “efficiency”56% 59%

Thus, although treatment redox efficiency is affected by free thiolconcentration, temperature and time, it can be effectively optimized andperformed over a wide range of variables. The treatment protocols canalso be performed on both small and large scale with reproducibility.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

1. A method comprising: contacting a preparation of a recombinantsoluble form of a p75 TNF-receptor that has been produced by mammaliancells with a reduction/oxidation coupling reagent, at a pH of about 7 toabout 11, and isolating a fraction of the preparation of the recombinantsoluble form of the p75 TNF-receptor with a desired conformation,wherein the desired conformation has a higher binding affinity than anundesired conformation for a cognate ligand of the p75 TNF-receptor. 2.The method of claim 1 wherein the recombinant soluble form of the p75TNF-receptor contains at least two domains.
 3. The method of claim 2wherein at least one domain of the recombinant soluble form of the p75TNF-receptor has a stable conformation, and at least one domain of theprotein has an unstable conformation.
 4. The method of claim 1 whereinthe recombinant soluble form of the p75 TNF-receptor is a Fc fusionprotein.
 5. The method of claim 4 wherein the preparation of therecombinant soluble form of the p75 TNF-receptor has been purified froma Protein A or Protein G column.
 6. The method of claim 1 wherein the pHis from about 7 to about
 10. 7. The method of claim 6 wherein the pH isabout 7.6 to about 9.6.
 8. The method of claim 7, wherein the pH isabout 8.6.
 9. The method of claim 1 wherein the reduction/oxidationcoupling reagent comprises glutathione.
 10. The method of claim 9wherein the ratio of reduced glutathione to oxidized glutathione isabout 1:1 to about 100:1.
 11. The method of claim 1 wherein thereduction/oxidation coupling reagent comprises cysteine.
 12. The methodof claim 1 wherein the contacting step is performed for about 4 to about16 hours.
 13. The method of claim 1 wherein the contacting step isperformed at about 25° C.
 14. The method of claim 1 wherein thecontacting step is performed at about 4° C.
 15. The method of claim 1wherein the contacting step is quenched by acidification.
 16. The methodof claim 1 wherein the isolating step comprises one or morechromatography steps.
 17. The method of claim 1 wherein the proteinconcentration of the recombinant soluble form of the p75 TNF-receptor isfrom about 0.5 to about 10 mg/ml.
 18. The method of claim 1 wherein theratio of reducing thiols in the reduction/oxidation coupling reagent todisulfide bonds in the protein is about 320:1 to about 64,000:1(reducing thiols: disulfide bond).
 19. The method of claim 1 furthercomprising formulating the fraction of the preparation of therecombinant soluble form of the p75 TNF-receptor with the desiredconformation in a sterile bulk form.
 20. The method of claim 1 furthercomprising formulating the fraction of the preparation of therecombinant soluble form of the p75 TNF-receptor with the desiredconformation in a sterile unit dose form.
 21. The method of claim 1wherein the desired conformation has a higher binding affinity for aTNF.
 22. The method of claim 21 wherein the TNF is TNF-alpha.
 23. Themethod of claim 1 wherein the contacting step is performed in a solutionessentially free of chaotrope.
 24. A method of promoting a desiredconformation of a glycosylated recombinant soluble form of a p75TNF-receptor, the method comprising contacting a preparation of theglycosylated recombinant soluble form of the p75 TNF-receptor thatcontains a mixture of at least two configurational isomers of theglycosylated recombinant soluble form of the p75 TNF-receptor with areduction/oxidation coupling reagent for a time sufficient to increasethe relative proportion of the desired configurational isomer anddetermining the relative proportion of the desired configurationalisomer in the mixture, wherein the desired configurational isomer has ahigher binding affinity than an undesired configurational isomer for acognate ligand of the p75 TNF-receptor.
 25. The method of claim 24wherein the glycosylated recombinant soluble form of the p75TNF-receptor contains at least two domains.
 26. The method of claim 25wherein at least one domain of the glycosylated recombinant soluble formof the p75 TNF-receptor has a stable conformation, and at least onedomain of the glycosylated recombinant soluble form of the p75TNF-receptor has an unstable conformation.
 27. The method of claim 24wherein the glycosylated recombinant soluble form of the p75TNF-receptor is a Fc fusion protein.
 28. The method of claim 27 whereinthe preparation of the glycosylated recombinant soluble form of the p75TNF-receptor has been purified from a Protein A or Protein G column. 29.The method of claim 24 wherein the pH is from about 7 to about
 10. 30.The method of claim 29 wherein the pH is about 8.6.
 31. The method ofclaim 24 wherein the reduction/oxidation coupling reagent is selectedfrom the group consisting of glutathione, cysteine, DTT(dithiothreitol), 2-mercaptoethanol and dithionitrobenzoate.
 32. Themethod of claim 31 wherein the reduction/oxidation coupling reagentcomprises reduced glutathione.
 33. The method of claim 32 wherein thereduced glutathione is at a concentration of about 1 mM to about 10 mM.34. The method of claim 31 wherein the reduction/oxidation couplingreagent comprises reduced cysteine.
 35. The method of claim 31 whereinthe ratio of reducing thiols in the reduction/oxidation coupling reagentto disulfide bonds in the protein is about 320:1 to about 64,000:1(reducing thiols: disulfide bond).
 36. The method of claim 24 whereinthe protein concentration is from about 0.5 to about 10 mg/ml.
 37. Themethod of claim 24 wherein the contacting step is performed for about 4to about 16 hours.
 38. The method of claim 24 wherein the contactingstep is performed at about 25° C.
 39. The method of claim 24 wherein thecontacting step is performed at about 4° C.
 40. The method of claim 24wherein the contacting step is quenched by acidification.
 41. The methodof claim 24 wherein the determining step comprises one or morechromatography steps.
 42. The method of claim 24 wherein the determiningstep comprises a binding reaction.
 43. The method of claim 24 comprisingisolating a fraction of the preparation of the glycosylated recombinantsoluble fonn of the p75 TNF-receptor with the desired configurationalisomer.
 44. The method of claim 43 comprising formulating the desiredconfigurational isomer in a sterile unit dose form.
 45. The method ofclaim 24 wherein the desired configurational isomer has a higher bindingaffinity for a TNF.
 46. The method of claim 45 wherein the TNF isTNF-alpha.
 47. The method of claim 24 wherein the contacting step isperformed in a solution essentially free of chaotrope.
 48. A methodcomprising formulating into sterile unit dose form a recombinant solubleform of the p75 TNF-receptor that has been produced by mammalian cells,contacted with a reduction/oxidation coupling reagent, and isolated fromthe fraction of the protein with an undesired conformation, wherein theundesired conformation has a lower binding affinity for a cognate ligandof the p75 TNF-receptor.
 49. The method of claim 48 wherein thecontacting step has been performed in a solution essentially free ofchaotrope.